Skip to main content
SpringerLink
Log in

Analysis of Methanogen Diversity in the Rumen Using Temporal Temperature Gradient Gel Electrophoresis: Identification of Uncultured Methanogens

  • Published:
Microbial Ecology Aims and scope Submit manuscript

Abstract

A temporal temperature gradient gel electrophoresis (TTGE) method was developed to determine the diversity of methanogen populations in the rumen. Tests with amplicons from genomic DNA from 12 cultured methanogens showed single bands for all strains, with only two showing apparently comigrating bands. Fingerprints of methanogen populations were analyzed from DNA extracted from rumen contents from two cattle and four sheep grazing pasture. For one sheep, dilution cultures selective for methanogens were grown and the culturable methanogens in each successive dilution examined by TTGE. A total of 66 methanogen sequences were retrieved from bands in fingerprints and analyzed to reveal the presence of methanogens belonging to the Methanobacteriales, the Methanosarcinales, and to an uncultured archaeal lineage. Twenty-four sequences were most similar to Methanobrevibacter ruminantium, five to Methanobrevibacter smithii, four to Methanosphaera stadtmanae, and for three, the nearest match was Methanimicrococcus blatticola. The remaining 30 sequences did not cluster with sequences from cultured archaea, but when combined with published novel sequences from clone libraries formed a monophyletic lineage within the Euryarchaeota, which contained two previously unrecognized clusters. The TTGE bands from this lineage showed that the uncultured methanogens had significant population densities in each of the six rumen samples examined. In cultures of dilutions from one rumen sample, TTGE examination revealed these methanogens at a level of at least 105 g−1. Band intensities from low-dilution cultures indicated that these methanogens were present at similar densities to Methanobrevibacter ruminantium-like methanogens, the sole culturable methanogens in high dilutions (106–10−10 g−1). It is suggested that the uncultured methanogens together with Methanobrevibacter spp. may be the predominant methanogens in the rumen. The TTGE method presented in this article provides a new opportunity for characterizing methanogen populations in the rumen microbial ecosystem.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4

Similar content being viewed by others

References

  1. Boadi, D, Benchaar, C, Chiquette, J, Masse, D (2004) Mitigation strategies to reduce enteric methane emissions from dairy cows: update review. Can J Anim Sci 84: 319–335

    Google Scholar 

  2. Brookman, JL, Nicholson, MJ (2005) Molecular fingerprinting techniques for genotypic analysis of pure cultures and microbial communities—anaerobic fungal populations. In: Makkar, HPS, McSweeney, CS (Eds.) Methods in Gut Microbial Ecology for Ruminants, Springer, Dordrecht, The Netherlands, pp 139–150

    Chapter  Google Scholar 

  3. Cole, JR, Chai, B, Marsh, TL, Farris, RJ, Wang, Q, Kulam, SA, Chandra, S, McGarrell, DM, Schmidt, TM, Garrity, GM, Tiedje, JM (2003) The Ribosomal Database Project (RDP-II): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy. Nucleic Acids Res 31: 442–443

    Article  PubMed  CAS  Google Scholar 

  4. Dojka, MA, Hugenholtz, P, Haack, SK, Pace, NR (1998) Microbial diversity in a hydrocarbon- and chlorinated-solvent-contaminated aquifer undergoing intrinsic bioremediation. Appl Environ Microbiol 64: 3869–3877

    PubMed  CAS  Google Scholar 

  5. Earl, J, Pickup, RW, Ritchie, DA, Edwards, C (2005) Development of temporal temperature gradient electrophoresis for characterising methanogen diversity. Microb Ecol 50: 327–336

    Article  PubMed  CAS  Google Scholar 

  6. Etokebe, GE, Spurkland, A (2000) Method for avoiding PCR-inhibiting contaminants when eluting DNA from polyacrylamide gels. Biotechniques 29: 694–696

    PubMed  CAS  Google Scholar 

  7. Felsenstein, J (1982) Numerical method for inferring taxonomic trees. Quant Rev Biol 27: 44–57

    Google Scholar 

  8. Garcia, JL, Patel, BKC, Ollivier, B (2000) Taxonomic phylogenetic and ecological diversity of methanogenic Archaea. Anaerobe 6: 205–226

    Article  PubMed  CAS  Google Scholar 

  9. Godon, JJ, Zumstein, E, Dabert, P, Habouzit, F, Moletta, R (1997) Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis. Appl Environ Microbiol 63: 2802–2813

    PubMed  CAS  Google Scholar 

  10. Hara, K, Shinzato, N, Seo, M, Oshima, T, Yamagishi, A (2002) Phylogenetic analysis of symbiotic archaea living in the gut of xylophagous cockroaches. Microbes Environ 7: 185–190

    Article  Google Scholar 

  11. Jarvis, GN, Strompl, C, Burgess, DM, Skillman, LC, Moore, ERB, Joblin, KN (2000) Isolation and identification of ruminal methanogens from grazing cattle. Curr Microbiol 40: 327–332

    Article  PubMed  CAS  Google Scholar 

  12. Joblin, KN, Naylor, GE, Williams, AG (1990) Effect of Methanobrevibacter smithii on xylanolytic activity of anaerobic ruminal fungi. Appl Environ Microbiol 56: 2287–2295

    PubMed  CAS  Google Scholar 

  13. Joblin, KN (1996) Options for reducing methane emissions from ruminants in New Zealand and Australia. In: Bouma, WJ, Pearman, GI, Manning, MR (Eds.) Greenhouse: Coping with Climate Change, CSIRO Publishing, Collingwood, Australia, pp 437–449

    Google Scholar 

  14. Joblin, KN (2005) Methanogenic archaea. In: Makkar, HPS, McSweeney, CS (Eds.) Methods in Gut Microbial Ecology for Ruminants, Springer, Dordrecht, The Netherlands, pp 47–53

    Chapter  Google Scholar 

  15. Kocherginskaya, SA, Aminov, RI, White, BA (2001) Analysis of the rumen bacterial diversity under two different diet conditions using denaturing gradient gel electrophoresis, random sequencing, and statistical ecology approaches. Anaerobe 7: 119–134

    Article  CAS  Google Scholar 

  16. Kocherginskaya, SA, Cann, IKO, Mackie, RI (2005) Denaturing gradient gel electrophoresis. In: Makkar, HPS, McSweeney, CS (Eds.) Methods in Gut Microbial Ecology for Ruminants, Springer, Dordrecht, The Netherlands, pp 119–128

    Chapter  Google Scholar 

  17. Lin, C, Raskin, L, Stahl, DA (1997) Microbial community structure in gastrointestinal tracts of domestic animals: comparative analyses using rRNA-targeted oligonucleotide probes. FEMS Microbiol Ecol 22: 281–294

    Article  CAS  Google Scholar 

  18. Ludwig, W, Strunk, O, Westram, R, Richter, L, Meier, H, Yadhukumar, Buchner, A, Lai, T, Steppi, S, Jobb, G, Forster, W, Brettske, I, Gerber, S, Ginhart, AW, Gross, O, Grumann, S, Hermann, S, Jost, R, Konig, A, Liss, T, Lussmann, R, May, M, Nonhoff, B, Reichel, B, Strehlow, R, Stamatakis, A, Stuckmann, N, Vilbig, A, Lenke, M, Ludwig, T, Bode, A, Schleifer, KH (2004) ARB: a software environment for sequence data. Nucleic Acids Res 32: 1363–1371

    Article  PubMed  CAS  Google Scholar 

  19. Miller TL (1995) Ecology of methane production and hydrogen sinks in the rumen. In: Engelhardt, WV, Leonhard-Marek, S, Breves, G, Giesecke, D (Eds.) Ruminant Physiology: Digestion, Metabolism, Growth and Reproduction, Ferdinand Enke Verlag Stuttgart, pp 317–331

  20. Paynter, MJB, Hungate, RE (1968) Characterisation of Methanobacterium mobilis, sp.n., isolated from the bovine rumen. J Bacteriol 95: 1943–1951

    PubMed  CAS  Google Scholar 

  21. Regensbogenova, M, Pristas, P, Javorsky, P, Moon-van der Staay, SY, van der Staay, GW, Hackstein, JH, Newbold, CJ, McEwan, NR (2004) Assessment of ciliates in the sheep rumen by DGGE. Lett Appl Microbiol 39: 144–147

    Article  PubMed  CAS  Google Scholar 

  22. Robertson, CE, Harris, JK, Spear, JR, Pace, NR (2005) Phylogenetic diversity and ecology of environmental Archaea. Curr Opin Microbiol 8: 638–642

    Article  PubMed  CAS  Google Scholar 

  23. Sharp, R, Ziemer, CJ, Stern, MD, Stahl, DA (1998) Taxon-specific associations between protozoal and methanogen populations in the rumen and a model rumen system. FEMS Microbiol Ecol 26: 71–78

    Article  CAS  Google Scholar 

  24. Skillman, LC, Evans, PN, Joblin, KN (2004) 16S ribosomal DNA-directed PCR primers for ruminal methanogens and identification of methanogens colonising young lambs. Anaerobe 10: 277–285

    Article  PubMed  CAS  Google Scholar 

  25. Skillman, LC, Evans, PN, Strompl, C, Joblin, KN (2006) 16S rDNA directed PCR primers and detection of methanogens in the bovine rumen. Lett Appl Microbiol 42: 222–228

    Article  PubMed  CAS  Google Scholar 

  26. Smith, PH, Hungate, RE (1958) Isolation and characterization of Methanobacterium ruminantium n. sp. J Bacteriol 75: 715–718

    Google Scholar 

  27. Soliva, CR, Meile, L, Cieslak, A, Kreuzer, M, Machmuller, A (2004) Myristic acid supports the immediate inhibitory effect of lauric acid on ruminal methanogens and methane release. Anaerobe 10: 269–276

    Article  PubMed  CAS  Google Scholar 

  28. Sprenger, WW, van Belzen, MC, Rosenberg, J, Hackstein, JH, Keltjens, JT (2000) Methanomicrococcus blatticola gen. nov., sp. nov., a methanol- and methylamine-reducing methanogen from the hindgut of the cockroach Periplaneta americana. Int J Syst Evol Microbiol 50(6): 1989–1999

    PubMed  CAS  Google Scholar 

  29. Tajima, K, Nagamine, T, Matsui, H, Nakamura, M, Aminov, RI (2001) Phylogenetic analysis of archaeal 16S rRNA libraries from the rumen suggests the existence of a novel group of archaea not associated with known methanogens. FEMS Microbiol Lett 200: 67–72

    Article  PubMed  CAS  Google Scholar 

  30. von Wintzingerode, F, Gobel, UB, Stackebrandt, E (1997) Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev 21: 213–229

    Article  Google Scholar 

  31. Watanabe, T, Asakawa, S, Nakamura, A, Nagaoka, K, Kimura, M (2004) DGGE method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil. FEMS Microbiol Lett 232: 153–163

    Article  PubMed  CAS  Google Scholar 

  32. Whitford, MF, Teather, RM, Forster, RJ (2001) Phylogenetic analysis of methanogens from the bovine rumen. BMC Microbiol 1: 5

    Article  PubMed  CAS  Google Scholar 

  33. Wright, AD, Williams, AJ, Winder, B, Christophersen, CT, Rodgers, SL, Smith, KD (2004) Molecular diversity of rumen methanogens from sheep in Western Australia. Appl Environ Microbiol 70: 1263–1270

    Article  PubMed  CAS  Google Scholar 

  34. Wright, AD, Toovey, AF, Pimm, CL (2006) Molecular identification of methanogenic archaea from sheep in Queensland, Australia reveal more uncultured novel archaea. Anaerobe 12: 134–139

    Article  PubMed  CAS  Google Scholar 

  35. Yanagita, K, Kamagata, Y, Kawaharasaki, M, Suzuki, T, Nakamura, Y, Minato, H (2000) Phylogenetic analysis of methanogens in sheep rumen ecosystem and detection of Methanomicrobium mobile by fluorescence in situ hybridization. Biosci Biotechnol Biochem 64: 1737–1742

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We gratefully acknowledge the Pastoral Greenhouse Gas Research Consortium of New Zealand for funding and support, and thank Ron Ronimus for comments on the manuscript.

Author information

Author notes

  1. Matthew J. Nicholson

    Present address: Institute of Molecular Biosciences, Massey University, Palmerston North, New Zealand

Authors and Affiliations

  1. Grasslands Reseach Centre, AgResearch, Private Bag 11008, Palmerston North, New Zealand

    Matthew J. Nicholson, Paul N. Evans & Keith N. Joblin

Authors
  1. Matthew J. Nicholson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paul N. Evans
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Keith N. Joblin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Keith N. Joblin.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nicholson, M.J., Evans, P.N. & Joblin, K.N. Analysis of Methanogen Diversity in the Rumen Using Temporal Temperature Gradient Gel Electrophoresis: Identification of Uncultured Methanogens. Microb Ecol 54, 141–150 (2007). https://doi.org/10.1007/s00248-006-9182-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00248-006-9182-1

Keywords

Search

Navigation